Hypersusceptibility to Substrate Analogs Conferred by Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

0022-538X/06/$08.00

⫹

0

doi:10.1128/JVI.00322-06

Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Hypersusceptibility to Substrate Analogs Conferred by Mutations in

Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Robert A. Smith,* Donovan J. Anderson, and Bradley D. Preston

Department of Pathology, University of Washington, Seattle, Washington 98195

Received 14 February 2006/Accepted 29 April 2006

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs

(A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming

nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an

important determinant of substrate specificity. To examine the functional role of this region, we performed

“random scanning mutagenesis” of 11 motif B residues and screened replication-competent mutants for

altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region

conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue

Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was

also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible

mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in

vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B

confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B

influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of

polymerization.

Conversion of viral RNA to double-stranded DNA by

re-verse transcriptase (RT) is a defining step in the retroviral life

cycle (8) and a key target of therapy for human

immunodefi-ciency virus type 1 (HIV-1) infection (17). The 66-kilodalton

subunit of HIV-1 RT contains four motifs (A, B, C, and D)

that are similarly arranged in all known structures of

replica-tive DNA and RNA polymerases (21). Three additional

struc-tural elements, motifs E and F and premotif A, are also

conserved among RTs and viral RNA-dependent RNA

poly-merases (4, 21, 45, 76). Together, motifs A, B, C, and F and

premotif A form a closely packed protein framework that

po-sitions the templating nucleotide, the primer terminus, and

incoming deoxynucleoside triphosphate (dNTP) at the RT

ac-tive site (Fig. 1A). Amino acid substitutions within this

con-served core can affect dNTP insertion fidelity, susceptibility to

nucleoside analogs, and/or discrimination against

ribonucleo-side triphosphates (rNTPs) during DNA synthesis (40, 49, 64,

72). Thus, these motifs influence the stringency and specificity

of substrate incorporation by RT.

Motif B is of particular interest because of its central

posi-tion in the RT core structure (Fig. 1) (12, 24). Motif B contacts

the template strand, the incoming dNTP, and each of the other

motifs in the core structure (premotif A and motifs A, C, and

F) (Fig. 1A), including residues within these other motifs that

are known to affect dNTP substrate recognition (Fig. 1B) (12,

24). The importance of motif B in substrate selection is evident

from studies of HIV-1 mutants resistant to nucleoside analogs

(49, 72). Two amino acid substitutions in motif B are

associ-ated with resistance to chain-terminating inhibitors: Q151M

and P157S (Fig. 1B). The Q151M replacement confers

low-level resistance to 3

⬘

-azido-3

⬘

-deoxythymidine (zidovudine

[AZT]), 2

⬘

,3

⬘

-dideoxyinosine (didanosine [ddI]), and 2

⬘

,3

⬘

-di-dehydro-3

⬘

-deoxythymidine (stavudine [d4T]) (25, 36). The

ad-dition of mutations at RT positions 62, 75, 77, and 116 in

combination with Q151M substantially increases the level of

resistance to these drugs both in vitro and in patients receiving

antiviral therapy (25, 36). The P157S mutation, originally

ob-served in a drug-resistant isolate of feline immunodeficiency

virus, confers resistance to (

⫺

)-

␤

-2

⬘

,3

⬘

-dideoxy-3

⬘

-thiacytidine

(lamivudine [3TC]) in both feline immunodeficiency virus and

HIV-1 (61, 62). Mutation P157S or P157A is occasionally

ob-served in RT sequences from patients receiving nucleoside

analog therapy (15, 43, 46, 52).

Additional evidence for the role of motif B in substrate

selectivity comes from biochemical studies of HIV-1 RT

mu-tants. Specific substitutions at position Q151 affect nucleotide

insertion fidelity, discrimination against rNTPs, and the

incor-poration of nucleotide analogs (10, 23, 28, 33, 54, 74).

Replace-ments at motif B residues V148, W153, K154, P157, and F160

also affect fidelity and/or analog incorporation in cell-free

poly-merase assays (11, 18, 29, 33, 56, 57, 74). However, most of the

RT mutants examined in these experiments exhibit significant

reductions in catalytic activity and are therefore unlikely to

support HIV-1 replication (13, 48). Thus, the importance of

motif B for substrate analog susceptibility and accurate DNA

synthesis during viral replication remains largely unexplored.

To this end, we used “random scanning mutagenesis” to

construct pools of HIV-1 variants, and we subjected these

pools to a single passage in culture to identify motif B

muta-tions that preserve viral replication capacity. Individual

vari-ants were then screened for altered sensitivity to nucleoside

and pyrophosphate analogs. Our results show that many motif

B residues influence substrate analog susceptibility and that

* Corresponding author. Mailing address: University of Washington,

Department of Pathology, K-084 HSB, Box 357705, 1959 NE Pacific

St., Seattle, WA 98195. Phone: (206) 221-5650. Fax: (206) 543-3967.

E-mail: [email protected].

7169

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specific substitutions in this region confer hypersusceptibility

to structurally diverse nucleoside analog inhibitors. The

phe-notypes exhibited by these mutants suggest that motif B

influ-ences both the substrate selectivity and the primer unblocking

activity of HIV-1 RT.

MATERIALS AND METHODS

Inhibitors.RT inhibitors ddI, d4T, and phosphonoformic acid (foscarnet [PFA]) were purchased from Sigma-Aldrich Co., St. Louis, Mo. Inhibitors (R )-9-(2-phosphonylmethoxypropyl)adenine (tenofovir [PMPA]) and (1S,4R )-4-[2-ami-no-6-(cyclopropyl-amino)-9H-purin-9-yl]-2-cyclopentene-1-methanol (abacavir [ABC]) were obtained from Moravek Biochemicals Inc., Brea, Calif. AZT was purchased from Moravek and Sigma-Aldrich. 3TC was kindly provided by Ray-mond Schinazi of Emory University or purchased from Moravek.

Plasmids, cells, and virus.All mutant strains and random virus pools were derived from a modified version of the full-length pR9 HIV-1 clone (65) that lacks an ApaI site in the plasmid backbone (kindly provided by Uta von Schwedler, University of Utah). pBSpolwas created by subcloning an ApaI/ EcoRI fragment of pR9 (HIV-1NL4-3, nucleotides 2010 to 5743) into pBluescript

II KS(⫺) (Stratagene, La Jolla, Calif.). pBSpolam(containing an amber stop

codon substitution at RT position 154) and pBSpolClaI(containing the insertion

GATCGAT at RT codon 152 [ClaI site underlined]) were generated from pBSpolby site-directed mutagenesis (Muta-Gene phagemid mutagenesis kit; Bio-Rad Laboratories, Hercules, Calif.). pR9⌬polwas created from pR9 by replacing the ApaI/EcoRI fragment with an ApaI-ATCGATGCGGCCGC -EcoRI synthetic linker (unique ClaI and NotI sites are underlined and italicized, respectively).

293tsA1609neo(293T) (51) and HeLa–CD4–LTR–␤-galactosidase (HeLa-P4)

(5) cells were cultured (37°C, 5% CO2) in Dulbecco’s modified Eagle’s medium

(DMEM; Invitrogen Corp., Carlsbad, Calif.) supplemented with 4 mML -glu-tamine, 50 U/ml penicillin, 50␮g/ml streptomycin, and 10% fetal bovine serum (HyClone, Logan, Utah).

Random-scanning mutagenesis.Mutant plasmid libraries containing random substitutions at individual codons in RT motif B were constructed by oligonu-cleotide-mediated mutagenesis using pBSpolam(for G152, K154, P157, and Q161

mutant pools) or pBSpolClaI(for the remaining mutant pools) as template DNA.

The oligonucleotides used to generate single-codon random mutants (Operon, Alameda, Calif.) spanned HIV-1NL4-3nucleotides 2971 to 3030 for pools

ran-domized at sites V148 to Q151, nucleotides 2982 to 3029 for G152, nucleotides 2977 to 3034 for W153 and G155, nucleotides 2991 to 3030 for K154, nucleotides 2982 to 3038 for S156, and nucleotides 3002 to 3043 for P157 and Q161.

Nucle-otide sequences at each randomized site were as follows: V148, HNN/NVN; L149, NVN/RNN; P150, NDN/DNN; Q151, NBN/DNN; G152, HNN/HHN/ NHN; W153, NNH; K154, BNN/BNY/NNY; G155, HNN/NHN; S156, NWN/ SNN/VNR; P157, NDN/DNN; and Q161, NBN/DNN (where N is A/T/G/C, D is A/G/T, B is C/G/T, H is A/C/T, V is A/C/G, R is A/G, Y is C/T, and S is C/G). These resulted in the exclusion of wild-type amino acids at each target codon and also excluded variants L149F, Q151H, K154M, S156C, S156W, and Q161H from their respective random pools. Products from the mutagenesis reactions were electroporated into ElectroMAX DH10BEscherichia coli(Gibco BRL), plas-mids were isolated from pools of⬎104

independent transformants, and ApaI/ EcoRI fragments from the pools were cloned into pR9⌬pol. The resulting full-length pR9 mutant libraries were purified from pools of⬎104 _independent

transformants using the Endo-Free maxiprep kit (QIAGEN Inc., Valencia, Calif.).

Transfections.To prepare wild-type virus stocks and random virus pools, CaPO4-pR9 DNA coprecipitates were prepared with 10␮g plasmid DNA as

described previously (6), except that the 2⫻BBS buffer was replaced with 2⫻ HEPES-buffered saline (270 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4· 2H2O,

11 mM dextrose, 40 mM HEPES, pH 7.05). 293T cultures were seeded into 10-cm plates and grown to approximately 25% confluence prior to the addition of CaPO4-DNA coprecipitates. Following a 3-hour incubation, culture

superna-tants were aspirated, 2 ml of 10% glycerol in phosphate-buffered saline (PBS) was added to each plate, and the cells were incubated at 37°C for 2 min. Cells were then rinsed twice with 6 ml of PBS, 10 ml of DMEM was added to each plate, and the cultures were returned to the incubator. Culture supernatants were harvested at approximately 42 h after transfection, filtered through 0.4-␮m sy-ringe filters, and stored in 1-ml aliquots in the vapor phase of liquid nitrogen for subsequent analysis. Stocks of individual HIV-1 mutants were produced by transfection of 293T cells as described above, except that the glycerol shock step was omitted. Cultures were instead treated with chloroquine at a final concen-tration of 25␮M in DMEM immediately before the DNA-CaPO4mixtures were

added. Supernatants were aspirated and replaced with fresh medium 1 day later and then harvested and frozen on the following day as described above. The titers of wild-type stocks produced by either protocol typically ranged from 2⫻ 105

to 5⫻105

focus forming units (FFU)/ml for frozen stocks and 1⫻106

to 4⫻ 106

FFU/ml for fresh preparations.

HeLa-P4 passage protocol.HeLa-P4 cells were seeded at 105_{cells per 10-cm}

plate and infected the next day with 104_{FFU in 2 ml DMEM containing 20␮g/ml}

DEAE-dextran (Sigma). After incubation at 37°C for 2 to 4 h, an additional 6 ml of DMEM was added to each plate, and incubation was continued overnight. The monolayers were then washed three times with 6 ml of PBS, and the cultures were replenished with 8 ml fresh medium, which was changed again after 2 days

FIG. 1. Relationship of motif B to other structural components in the ternary complex of HIV-1 RT (Protein Data Bank entry 1RTD) (24).

(A) Surface representation of conserved structural motifs in the polymerase domain. Amino acids 107 to 118 (motif A), 147 to 169 (motif B), and

180 to 189 (motif C) are shown, as assigned by Poch et al. (53). Residues 102 to 106 (motif A), 143 to 146 (motif B), and 190 (motif C) are omitted

for clarity. Boundaries for motif F (residues 64 to 75) are based on recent alignments of viral RNA-dependent RNA polymerases (4, 76). The

region designated “premotif A” (residues 75 to 91) was originally identified in an alignment of HIV-1 RT with negative-stranded RNA virus

polymerases (45). (B) Location of several motif B amino acids and other residues discussed in the text. Motif F and premotif A have been removed

for clarity. Amino acid substitutions at residues Q151 and P157 (labeled in red) are known to confer resistance to nucleoside analogs (25, 36, 61).

Residues Y115 and M184 are key determinants of dNTP substrate selectivity in motifs A and C, respectively (40, 64). Mg

ions coordinated at

the active site are shown as small black spheres. These views were produced using MacPyMOL version 0.95 (http://pymol.sourceforge.org).

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of incubation. Culture supernatants were harvested on the fifth day after infec-tion, passed through 0.4-␮m filters, and treated with 420 U DNase I (Worthing-ton Biochemical) at 37°C for 20 min after we added MgCl2to a final

concen-tration of 10 mM. Viral particles were concentrated by layering 1 ml of culture supernatant onto a 250-␮l cushion of 20% sucrose in phosphate-buffered saline, followed by centrifugation at 15,500⫻gfor 90 min at 4°C. Virion pellets were resuspended in 100␮l of lysis buffer (7 M urea, 0.35 M NaCl, 4 mM EDTA, 10 mM Tris-HCl, pH 7.5, 1% sodium dodecyl sulfate) and stored at⫺20°C for subsequent RNA extraction and RT-PCR amplification. The remaining uncon-centrated culture supernatants were frozen in 1-ml aliquots in the vapor phase of liquid nitrogen.

RT-PCR and DNA sequencing.RT-PCR was performed in thin-walled tubes (Robbins), using the Access RT-PCR system (Promega), in 50-␮l reaction mix-tures containing 200␮M of each dNTP, 1 mM MgSO4, 5 U avian myeloblastosis

virus RT, 5 UTflDNA polymerase, 10␮l of 5⫻avian myeloblastosis virus-Tfl

buffer, 50 pmol each of primers H3 and BH1 (see below), and 1␮l of extracted viral RNA. Primers BH1 (5⬘-TATGGATCCCTTTTAGAATCTCCCTGTTTTC TGCC-3⬘; BamHI site underlined) and H3 (5⬘-AGTCAAGCTTGGATGGCCC AAAAGTTAAACAATGGCC-3⬘; HindIII site underlined) amplified a 0.8-kb fragment corresponding to nucleotides 2597 to 3483 of HIV-1NL4-3.

Thermocy-cling conditions in an MJ Research PTC-100 thermocycler were as follows: 48°C for 45 min, 94°C for 2 min, then 40 cycles of 94°C for 30 s, 60°C for 1 min, and 68°C for 2 min, and ending with 68°C for 7 min. Control reactions lacking RT were performed and analyzed in parallel to ensure that amplification was RNA dependent. When required, viral RNA samples were subjected to a second DNase I treatment to remove contaminating plasmid or proviral DNA (see above). RT-PCR products were digested with BamHI and HindIII, ligated into double-digested pBluescript II KS(⫺), transformed intoE. coli, and spread on agar plates containing ampicillin. Individual colonies were randomly picked from these plates, and plasmids were isolated and sequenced using primer pNL1 (5⬘-GACTTCAGGAAGTATACTGC-3⬘) and BigDye Terminator chemistry (Applied Biosystems, Foster City, Calif.).

Infectivity assay.Single-round infectivities were determined using HeLa-P4 indicator cells as previously described (5, 61). Briefly, HeLa-P4 cells were seeded into 96-well plates at 0.5⫻104_{cells/well and infected the following day with}

serial dilutions of virus prepared in DMEM containing 20␮g/ml DEAE-dextran. After 40 h of growth, cultures were fixed and stained with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal; Promega, Madison, Wis.). In this time frame, infected cells appeared as isolated groups of two to five contiguous Lac⫹ (blue) cells, indicating that the majority of foci were derived from a single cycle of virus replication. Titers of Lac⫹foci were normalized against HIV-1 capsid p24 concentration (HIV-1 enzyme-linked immunosorbent assay; PerkinElmer, Boston, MA) to determine the infectivities of mutants relative to that of the wild-type virus. The infectivity of the wild-type pR9-derived HIV-1NL4-3was

880⫾180 FFU/ng p24 (mean⫾standard error) for frozen stocks and 4,500⫾ 1,100 FFU/ng p24 for fresh supernatants. A variant containing the RT-inactivat-ing D185A mutation served as a negative control and yielded 0.014⫾0.004 FFU/ng p24 from frozen stocks.

Inhibitor sensitivity assay.For measurements of inhibitor sensitivity, HeLa-P4 cells were seeded into 96-well plates at a density of 0.5⫻104

cells/well and incubated overnight. Culture wells were dosed with various concentrations of inhibitor on the following morning, and the plates were then returned to the incubator for an additional 3 hours. Immediately prior to infection, virus stocks were diluted to 4,000 FFU/ml in DMEM containing 20␮g/ml of DEAE-dextran. Supernatants in the microtiter plates were aspirated, and 25␮l of the virus dilution was added directly to the monolayer in each well. Plates were then returned to the incubator for 3 hours. After this time, an additional 175␮l medium was added to each well, a second dose of inhibitor was added (at the same concentration as the first dose), and incubation was continued for two more days. Culture monolayers were fixed and stained as described above, and Lac⫹ foci were counted. Control cultures incubated in the absence of substrate analogs typically yielded 70 to 250 foci/well. Concentrations of analog required to inhibit focus formation by 50% of the untreated control value (50% effective concen-tration [EC50]) were calculated by linear regression of the resulting

dose-re-sponse data.

RESULTS

Random scanning mutagenesis of motif B.

To examine the

role of motif B in substrate analog sensitivity, we first

gener-ated pools of HIV-1 mutants containing random single amino

acid replacements at each of 11 motif B residues (60). This

strategy is an extension of alanine scanning mutagenesis (9)

and is referred to here as “random scanning mutagenesis.” The

benefit of random scanning mutagenesis is that all 19 amino

acid substitutions are introduced at each position, thus

permit-ting the detection of functional changes that are not produced

by simple alanine substitutions.

We used two different strategies to optimize the yield of

random amino acid substitutions and minimize the proportion

of wild-type sequences at each target codon. In the first

ap-proach, we constructed a

pol

subclone that contained a lethal

amber stop codon at RT position K154 (pBSpol

). Mutagenic

oligonucleotides were used to correct this stop codon and

simultaneously introduce random substitutions at RT codon

G152, K154, P157, or Q161. Following mutagenesis, the

pol

genes were ligated into a

pol-deleted HIV-1 construct

(pR9

⌬

pol) to produce full-length HIV-1 plasmid libraries,

each randomized at a specific codon in RT motif B. Sequence

analyses of 180 individual pR9 clones generated by this method

confirmed that each library contained a diverse array of amino

acid substitutions at the targeted motif B position. Pools

ran-domized at codon G152, K154, P157, or Q161 contained 12, 16,

17, or 14 of 20 possible amino acid residues, respectively, at the

target codon. However, each pool also contained a substantial

number of nonmutant clones that retained the K154 amber

stop codon (50% in the K154, P157, and Q161 pools and 85%

in the G152 pool). Although these K154 amber products did

not interfere with subsequent experimental steps, sequencing

of large numbers of clones was required in order to

character-ize the diversity of each library.

To increase mutagenesis efficiency, we devised a second

strategy for random mutagenesis of the remaining seven target

residues in motif B. This method started with a

pol

subclone

containing a unique ClaI restriction site at position 152 of RT

(pBSpol

). Oligonucleotide primers restored the wild-type

sequence at codon 152 while simultaneously introducing

ran-dom mutations at position 148, 149, 150, 151, 153, 155, or 156

of RT. After subcloning into pR9

⌬

pol

and amplification in

E.

coli, the resulting full-length HIV-1 plasmid libraries were

en-riched for mutant clones by being digested with ClaI, followed

by a third round of

E. coli

amplification. Sequencing of

indi-vidual clones generated by this second strategy showed that the

final proportion of mutant plasmids in the resulting libraries

was

⬎

90% and that the diversity of these libraries was similar

to the diversity achieved using the initial pBSpol

-based

ap-proach (see above). Both mutagenesis strategies limited but

did not completely exclude wild-type clones, which were

present at frequencies of 5 to 12% in the plasmid libraries.

Selection of replication-competent mutants.

The full-length

mutant HIV-1 plasmid libraries were separately transfected

into 293T cells to produce mutant virus pools, each comprised

of variants with random replacements at a single codon in

motif B. Replication-competent viruses were selected from

these pools by subjecting the virus populations to a single 5-day

passage in HeLa-P4 cells (see Materials and Methods for

de-tails). Infectious mutants were then identified by sequencing

individual clones of RT-PCR products derived from the

result-ant HeLa-P4-passaged pools. Altogether, 52 different single

amino acid substitutions in motif B were detected following a

single passage in culture (Table 1). Eight of the 11 pools

(V148, L149, P150, Q151, K154, G155, P157, and Q161)

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yielded mutants with both conservative and nonconservative

replacements at the respective target codons. Two pools

(W153 and S156) yielded only a single variant, and only one

pool (G152) failed to produce infectious mutants.

To confirm that the variants identified in the

HeLa-P4-se-lected populations were viable, we performed two separate

analyses of viral replication. First, the K154, P157, and Q161

mutant virus pools were subjected to three additional passages

in culture, and the range of mutants present in these

subse-quent cultures was determined by sequencing 25 to 50 clones

from each passage interval. Of the 25 mutants identified in the

first passage of these mutant pools (Table 1), 23 were detected

in passage 2, 3, or 4 (60). Thus, the majority of mutants

ob-served after a single passage in culture were capable of

multi-ple cycles of replication. We also constructed 24 full-length

HIV-1 plasmid clones containing specific single amino acid

substitutions in motif B that were detected in the passage 1

supernatants. In most cases, the variants produced by these

clones retained

ⱖ

50% of wild-type infectivity (Fig. 2).

Alto-gether, 47 of the 52 mutants observed in the virus pools

fol-lowing a single passage in HeLa-P4 cells (Table 1) were

rep-lication competent, as evidenced by their infectivity as purified

clones (Fig. 2) and/or persistence in subsequent passages (60).

Residues throughout motif B affect nucleoside analog

sen-sitivity.

To assess the functional importance of motif B, we

initially measured the susceptibility of each

replication-compe-tent mutant shown in Fig. 2 to the cytidine nucleoside analog

3TC. Several mutations in motif B altered the sensitivity of the

virus to this analog (Fig. 3A and 4A). Overall, there was a

trend towards 3TC resistance among the variants analyzed

(Fig. 4A), although many substitutions were neutral and three

replacements (W153F, G155N, and G155Q) resulted in slight

hypersusceptibility to 3TC.

We also examined the susceptibility of each motif B mutant

to the thymidine analog AZT. As previously reported, the

Q151M mutation conferred moderate resistance to AZT, and

the Q151M/A62V/V75I/F77L/F116Y complex of mutations

conferred

⬎

50-fold resistance to the drug (25, 36). In contrast,

most of the other motif B mutants examined were

hypersus-ceptible to AZT (Fig. 3B and 4B). Specific substitutions at

positions V148, Q151, and G155 conferred 10- to 60-fold

in-creases in AZT sensitivity. Altogether, 20 of the 24 motif B

variants examined were hypersusceptible to AZT and/or

resis-tant to 3TC (Fig. 4). Specific mutations at codons V148, Q151,

and P157 conferred both AZT hypersusceptibility and 3TC

resistance.

We noted that a subset of AZT-hypersusceptible mutants

exhibited a substantial impairment in viral replication capacity

(Fig. 2). To further examine the relationship between

hyper-susceptibility and viral infectivity, we characterized the effects

of four specific mutations in the “primer grip” region of RT

(26). An F227A RT mutant was eightfold hypersensitive to

AZT and retained 27% of wild-type HIV-1 infectivity,

demon-strating that mutations outside of motif B can also confer AZT

hypersusceptibility. In contrast, variants W266F, W266Y, and

W266R exhibited 0.5, 27, and 92% of wild-type infectivity,

respectively, but

ⱕ

2-fold changes in AZT or 3TC sensitivity.

Similarly, the P150A replacement in motif B, which reduced

viral infectivity to 15% of the wild type, had no effect on AZT

sensitivity, while other variants that retained 80 to 100%

rep-lication capacity were

ⱖ

10-fold hypersusceptible to AZT (e.g.,

FIG. 2. Replication capacities of RT motif B mutants. 293T cells were transfected with the wild-type (WT) HIV-1 pR9 clone or with clones

containing specific substitutions in motif B. Titers in the resulting cultures were measured using HeLa-P4 cells and normalized to the concentration

of HIV-1 capsid p24 in the supernatant to determine the infectivity of each mutant relative to that of the wild-type virus. ND, not determined;

Q151M

⫹

4, multinucleoside-resistant mutant Q151M/A62V/V75I/F77L/F116Y (25).

*

, statistically different from the wild type by one-way analysis

of variance (

P

⬍

0.05).

TABLE 1. Amino acid substitutions observed in the mutant virus

pools following a single passage in HeLa-P4 cells

Positiona

Amino acid(s) (no. of clones)b

V148 ...

V

(

18

)

, I (1), S (2), C (3), T (1), R (1)

L149 ...

L

(

18

)

, I (6), T (1), M (2)

P150 ...

P

(

24

)

, A (2), K (1)

Q151... G (7), A (12), V (1), I (1), S (1), C (1), T (3), M (4) G152...G(19)

W153 ... W(25), F (1)

K154 ... G (10), A (2), V (7), L (5), S (6), C (4), T (9), R (1), N (1), W (1)

G155...G(11), A (4), V (1), L (1), S (1), C (1), M (2), N (3), Q (2) S156...S(20), A (25)

P157 ...P(18), G (13), A (2), L (1), S (2), C (4), T (3)

Q161...Q(8), G (8), A (4), V (3), L (2), S (3), T (1), M (3), E (4)

a_{Pools of mutants were produced and manipulated as independent virus} populations, each containing random mutations at the specified amino acid position in RT.

b_{RT-PCR products amplified from HeLa-P4 supernatants were cloned into a} plasmid vector and sequenced. Numbers in parentheses indicate the number of clones containing wild-type (bold italics) or mutant (roman) amino acids at the targeted RT codon. No wild-type clones were observed in random mutant pool Q151 or K154. Data are retabulated from reference 60.

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Q151A and Q161G) (Fig. 2 and 4B). Taken together, these

data indicate that AZT hypersusceptibility does not generally

correlate with reduced viral replication capacity.

Relationship between AZT hypersusceptibility and PFA

re-sistance.

Specific replacements at codons 88, 89, 90, 92, 156,

160, 161, and 164 of HIV-1 RT have previously been shown to

confer slight increases in AZT sensitivity (20, 39, 42, 67, 68).

These substitutions also confer resistance to PFA, an analog

that mimics the

␤

-

␥

pyrophosphate group of the incoming

dNTP substrate. To further examine this phenotypic

relation-ship, we measured the sensitivities of 14 different

AZT-hyper-susceptible motif B mutants to PFA (Fig. 5). We also included

the S156A variant in these experiments as a positive control

for PFA resistance (67, 68). Six of the variants (V148C,

V148R, V148S, P157G, P157S, and Q161G) were resistant

to PFA (Fig. 5A) and therefore fit the aforementioned

pat-tern of AZT hypersusceptibility and PFA resistance. As

previously reported, the S156A mutation conferred fourfold

resistance to PFA without significantly affecting viral

sensi-tivity to AZT (67, 68).

We also observed several variants that did not follow the

AZT-hypersusceptible, PFA-resistant pattern (Fig. 5). For

ex-ample, mutants Q151G, Q151C, G155N, and G155Q displayed

6- to 20-fold hypersusceptibility to AZT but were not

signifi-cantly resistant to PFA. In addition, the Q151V and Q151I

variants showed increased sensitivity to both AZT and PFA

(Fig. 5 and Table 2). These results demonstrate that the

AZT-hypersusceptible phenotype is not necessarily coupled to PFA

resistance.

Variants hypersusceptible to other nucleoside analogs.

Sev-eral substitutions at position Q151 resulted in 10-fold or

greater hypersusceptibility to AZT (Fig. 4B), suggesting that

this residue is particularly important for substrate analog

sen-sitivity. We therefore determined the response of the Q151

mutants to a panel of structurally diverse nucleoside analog

inhibitors (Table 2 and Fig. 6). Consistent with previous

re-ports, the Q151M mutation resulted in low-level resistance to

d4T and ddI, and Q151M combined with A62V, V75I, F77L,

and F116Y conferred higher levels of resistance to these two

analogs (Table 2) (25, 36). In contrast, replacement of glycine,

alanine, valine, isoleucine, or cysteine at position 151 increased

HIV-1 sensitivity to several of the nucleoside analogs tested.

Mutants Q151G and Q151C were 5- to 10-fold

hypersuscep-tible to d4T, PMPA, and ABC (Table 2). Variants Q151V and

Q151I were also 10-fold hypersusceptible to d4T and PMPA

but showed only marginal hypersusceptibility to ABC. The

Q151A mutation resulted in two- to threefold increases in d4T,

PMPA, and ABC sensitivities. With the exception of

methio-nine, each of the Q151 substitutions also conferred modest

hypersusceptibility to ddI, with EC

s two- to fivefold lower

than that of the wild type. Thus, several different substitutions

at Q151 conferred a multinucleoside-hypersusceptible

pheno-type (Fig. 6).

In addition to Q151 mutations, specific substitutions at other

motif B positions also conferred multinucleoside

hypersuscep-tibilty. Mutants V148R and G155Q, both of which were

hyper-susceptible to AZT (Fig. 4A), were also four- to sevenfold

hypersusceptible to PMPA (EC

s of 1.4

⫾

0.2

␮

M and 0.90

⫾

0.3

␮

M for V148R and G155Q, respectively, versus 5.9

⫾

1.0

␮

M for the wild-type virus). The G155Q variant also exhibited

three- to fivefold hypersusceptibility to d4T, ddI, and ABC but

no change in PFA sensitivity (data not shown).

In summary, substitutions at 9 of the 11 RT motif B positions

subjected to random mutagenesis altered viral susceptibility to

one or more nucleoside analogs, and several mutations

con-ferred altered sensitivity to the pyrophosphate analog PFA.

Thus, residues throughout RT motif B play important roles in

determining the substrate analog sensitivity of HIV-1.

FIG. 3. Representative dose-response data for the pyrimidine analogs 3TC (A) and AZT (B). Profiles for wild-type HIV-1 (dotted lines) and

several motif B variants (solid lines) are shown. Analog sensitivities were measured by quantitating the dose-dependent reduction of Lac

foci in

HeLa-P4 cells. The percentages of solvent-only control foci are plotted as a function of nucleoside analog concentration. Curves were generated

using a sigmoidal regression equation (GraphPad Prism 4 software package [44]). The results for each strain are from a single assay, with two

determinations of focus formation per drug concentration. These data are representative of the responses observed for each mutant in multiple

independent experiments.

F

, P157S;

E

, W153F;

■

, wild type;

䊐

, Q151A;

ƒ

, Q151V;

Œ

, Q151M/A62V/V75I/F77L/F116Y;

‚

, Q161G;

䉬

, Q151M;

䉫

, G155Q.

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DISCUSSION

The polymerase domain of HIV-1 RT contains structural

motifs that are conserved in all reverse transcriptases and

RNA-dependent viral polymerases (Fig. 1) (4, 21, 76). Amino

acid residues within these motifs affect the fidelity of DNA

synthesis and influence the sensitivity of HIV-1 to substrate

analog inhibitors (40, 49, 64, 72). Motif B is particularly

inter-esting because of its central position in the core polymerase

structure (Fig. 1). Although a few mutations in HIV-1 RT

motif B have been shown to confer drug resistance (25, 36, 39,

61, 67), the role of this structure in viral sensitivity to

nucleo-side and pyrophosphate analogs is largely unknown. To

exam-ine the functional importance of motif B in the context of

replicating virus, we used random scanning mutagenesis to

introduce random single amino acid replacements at 11 motif

B codons. We then identified the range of replacements that

preserve HIV-1 replication in culture (Table 1 and Fig. 2) and

screened a subset of these infectious mutants for altered

sen-sitivity to substrate analog inhibitors. The results demonstrate

that residues throughout the targeted region of motif B affect

viral susceptibility to substrate analogs (Fig. 3 to 5) and that

specific substitutions, particularly at residue Q151, confer a

multidrug-hypersusceptible phenotype (Table 2 and Fig. 6).

Previous efforts to identify determinants of dNTP selectivity

have primarily used conventional site-directed mutagenesis to

introduce single amino acid replacements in RT (40, 64).

FIG. 4. Susceptibilities of motif B mutants to 3TC (A) and AZT (B). Concentrations of nucleoside analog required to inhibit Lac

focus

formation in HeLa-P4 cells by 50% (EC

) were calculated by regression analyses of dose-response data (Fig. 3), as described in Materials and

Methods. The abscissa is set at the EC

for wild-type (WT) virus; bars above and below the abscissa represent analog resistance and

hypersus-ceptibility, respectively. Each bar represents the mean

⫾

standard error from at least three independent experiments.

*

, significantly different from

the wild type by one-way analysis of variance (

P

⬍

0.05);

**

, significantly different from the wild type by a paired

t

test (

P

⬍

0.05). ND, not

determined. Variants Q151M

⫹

4 (Q151M/A62V/V75I/F77L/F116Y) and M184V, which exhibit high-level resistance to AZT and 3TC, respectively,

served as positive controls in these assays (25, 55, 69).

FIG. 5. Sensitivities of AZT-hypersusceptible mutants to PFA.

EC

s for PFA (A) and AZT (B) were measured and graphed as

described in Materials and Methods and the legend to Fig. 3. Each bar

represents the mean

⫾

standard error from at least three independent

experiments.

*

, significantly different from the wild type (WT) by

one-way analysis of variance (

P

⬍

0.05).

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These variant RTs often exhibit severe impairments of

poly-merase function and are therefore unable to support viral

replication (13, 18, 23, 28, 48, 56, 57, 59, 74). Here, we used cell

culture to select replication-competent viruses from random

pools of HIV-1 RT mutants. A sampling of 24 culture-selected

mutants showed that most retained

ⱖ

50% of wild-type HIV-1

infectivity (Fig. 2). Thus, random scanning mutagenesis

cou-pled to selection in culture is a facile approach for identifying

catalytically active RT mutants. The strategies we used to

en-rich viable mutants and limit the proportion of wild-type

HIV-1 in the random virus pools can readily be applied to

other regions of the HIV-1 genome as well as other viruses for

which a full-length plasmid clone is available.

A notable finding from our study was the magnitude and

scope of substrate analog hypersusceptibility produced by

mu-tations in RT motif B. Substitutions at 8 of the 11 residues

analyzed increased the sensitivity of HIV-1 to one or more

inhibitors, and several variants displayed 10-fold or greater

analog hypersusceptibility (Fig. 4 and 5 and Table 2). Previous

studies of HIV-1 variants have reported low-level (i.e., two- to

fivefold) increases in viral sensitivity to nucleoside analogs (20,

39, 42, 61, 67, 68, 73), and replacements in conserved regions

of herpesvirus and bacteriophage

␾

29 DNA polymerases

oc-casionally confer low-level hypersusceptibility to substrate

an-alogs (3, 7, 14, 19, 38, 70). Our data demonstrate that single

amino acid substitutions in HIV-1 RT motif B can produce

large increases in inhibitor sensitivity (Fig. 3 to 5) while

pre-serving viral replication capacity (Fig. 2). We anticipate that

other conserved RT motifs play a similar role in substrate

analog susceptibility.

Our analysis of motif B suggests that nucleoside analog

hypersusceptibility results from multiple biochemical

mecha-nisms that are not necessarily mutually exclusive. First, a subset

of motif B mutations may affect hypersensitivity to AZT by

impairing the “primer unblocking” activity of RT (41). This

inference is supported by the observation that specific

muta-FIG. 6. Hypersusceptibility of Q151 mutants to substrate analog inhibitors. Data from Fig. 4 and 5 and Table 2 are plotted to illustrate the

change (

n

-fold) in EC

for each Q151 mutant relative to that of wild-type HIV-1. Values above and below the

x

axis are resistant and

hypersusceptible to drug, respectively. Horizontal bars indicate the median change (

n

-fold) in EC

for each analog for the entire set of Q151

mutants.

■

, Q151G;

䊐

, Q151M;

, Q151V;

Œ

, Q151A;

䉬

, Q151I;

F

, Q151C.

TABLE 2. Susceptibilities of Q151 RT mutants to substrate analogs

Virus

EC50(␮M)a

3TC AZT d4T ddI PMPA ABC PFA

Wild type

0.90

⫾

0.1 (1)

0.17

⫾

0.02 (1)

5.8

⫾

0.9 (1)

3.1

⫾

0.8 (1)

5.9

⫾

1 (1)

7.0

⫾

1 (1)

92

⫾

6 (1)

Q151G

0.79

⫾

0.3 (1)

0.0088

⫾

0.001 (0.1)

0.36

⫾

0.1 (0.1)

1.2

⫾

0.3 (0.4)

0.92

⫾

0.1 (0.2)

1.3

⫾

0.1 (0.2)

93

⫾

36 (1)

Q151A

3.4

⫾

0.5 (4)

0.013

⫾

0.003 (0.1)

2.8

⫾

2 (0.5)

1.1

⫾

0.1 (0.3)

1.8

⫾

0.2 (0.3)

2.0

⫾

0.5 (0.3)

62

⫾

8 (0.7)

Q151V

9.3

⫾

1 (10)

0.0028

⫾

0.001 (0.02)

0.58

⫾

0.2 (0.1)

1.4

⫾

0.1 (0.4)

0.36

⫾

0.1 (0.1)

3.4

⫾

0.5 (0.5)

6.7

⫾

0.9 (0.1)

Q151I

7.7

⫾

2 (9)

0.016

⫾

0.001 (0.1)

0.55

⫾

0.04 (0.1)

0.69

⫾

0.3 (0.2)

0.48

⫾

0.1 (0.1)

3.2

⫾

0.5 (0.5)

11

⫾

2.2 (0.1)

Q151C

1.5

⫾

0.6 (2)

0.012

⫾

0.002 (0.1)

0.30

⫾

0.05 (0.1)

0.73

⫾

0.2 (0.2)

0.46

⫾

0.1 (0.1)

0.71

⫾

0.2 (0.1)

150

⫾

130 (2)

Q151M

1.4

⫾

0.3 (2)

0.64

⫾

0.02 (4)

23

⫾

5 (4)

11

⫾

2 (3)

7.8

⫾

2 (1)

17

⫾

3 (2)

160

⫾

60 (2)

Q151M

⫹

4

6.3

⫾

2 (7)

⬎

10 (

⬎

100)

⬎

100 (

⬎

20)

39

⫾

5 (13)

18

⫾

1 (3)

56

⫾

18 (8)

ND

a

EC50s were obtained for HeLa-P4 cells as described in Materials and Methods. Numbers in parentheses indicate EC50s relative to that of the wild-type virus. Values

are the means⫾standard errors from three or more independent experiments. b

Multinucleoside-resistant mutant Q151M/A62V/V75I/F77L/F116Y (27, 37). c

ND, not determined.

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tions conferred both AZT hypersusceptibility and PFA

resis-tance (Fig. 5), a phenotypic pattern that has been correlated

with a loss of unblocking capacity (1, 42). Diminished primer

unblocking function may occur with or without compromised

RT polymerase activity, as suggested by the subset of

hyper-susceptible variants with reduced infectivity (Fig. 2 and 4).

Second, mutations in RT may confer nucleoside analog

hy-persusceptibilty by enhancing the efficiency of analog

incorpo-ration. Specific replacements at position Q151 increased the

sensitivity of HIV-1 to multiple substrate analogs (Table 2),

suggesting a direct effect on nucleotide selectivity (see below).

Other mutations in motif B are likely to impart nucleoside

analog hypersusceptibility indirectly by repositioning residues

Y115 and M184 (Fig. 1B), which are key determinants of

substrate specificity (40, 64). Taken together, our data suggest

that motif B influences both the efficiency of the primer

un-blocking reaction and the selectivity of nucleotide

incorpora-tion by HIV-1 RT. Thus, motif B likely contributes to

impor-tant enzyme-substrate interactions at multiple steps in the

catalytic cycle of polymerization.

Our results demonstrate that residue Q151 is particularly

important for substrate analog sensitivity (Table 2 and Fig. 6).

Substitutions at this position presumably affect the interaction

between RT and the incoming dNTP (Fig. 1) (24), thereby

directly influencing analog binding and/or polymerization (10).

It is well established that the Q151M mutation contributes to

nucleoside analog resistance in HIV-1 and simian

immunode-ficiency virus (25, 36, 71). Here, we show that other Q151

substitutions confer broad-spectrum hypersusceptibility to

structurally diverse nucleoside analogs, with up to a 60-fold

increase in analog sensitivity. Moreover, we show that a

sub-set of Q151 mutations also imparts hypersusceptibility to the

pyrophosphate analog PFA. These data suggest that specific

Q151 replacements in HIV-1 RT confer a general relaxation

of polymerase active-site stringency. Additional experiments

are required to examine the relationship between multidrug

hypersusceptibility and other aspects of RT substrate

selec-tivity, such as mispair formation and rNTP versus dNTP

discrimination.

Residues that are structurally analogous to Q151 of HIV-1

RT also influence nucleoside inhibitor sensitivity in other

DNA polymerases. In family A (E. coli

DNA polymerase

I-re-lated) enzymes, a conserved aromatic residue (phenylalanine

or tyrosine) that is positioned similarly to Q151 in the polymerase

active site strongly influences dNTP versus dideoxynucleoside

triphosphate selectivity (2, 35, 66). Substitutions at a structurally

equivalent asparagine residue in family B (mammalian DNA

polymerase

␣

-related) polymerases also affect fidelity and/or

nu-cleoside analog sensitivity (27, 31, 47). Taken together, these data

indicate that residues analogous to Q151 influence the inhibitor

sensitivities of polymerases from diverse organisms.

In patients receiving antiviral therapy, drug treatment

occa-sionally selects for variants that are resistant to one or more

components of the administered regimen but are

hypersuscep-tible to other inhibitors. For example, mutations that emerge

in vivo in response to 3TC, ddI, PFA, or certain nonnucleoside

reverse transcriptase inhibitors can confer hypersusceptibility

to AZT and “resensitize” AZT-resistant viruses (20, 30, 32, 42,

63, 68, 75). Increased viral sensitivity to protease inhibitors and

nonnucleoside reverse transcriptase inhibitors in clinical

iso-lates of HIV-1 has also been reported (16, 34, 37, 58). Our

analysis suggests that there are a number of RT mutations that

confer hypersusceptibility to AZT and other nucleoside

ana-logs. These mutations may contribute to observed differences

in drug sensitivity among “wild-type” HIV-1 isolates (22, 50)

and could potentially influence the efficacy of

nucleoside-con-taining antiretroviral regimens.

ACKNOWLEDGMENTS

We thank Tom North, Masanori Ogawa, and Tina Albertson for

critical reading of the manuscript and Crystal Pyrak for excellent

tech-nical assistance.

This work was supported by Public Health Service grants R01

AI34834 to B.D.P. and F32 AI10139 to R.A.S.

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